Infectious atypical pneumonia is referred to as Severe Acute Respiratory Syndrome (SARS). From the end of 2002 to the beginning of 2003, patients infected with SARS were initially found in the Province of Guangdong of China. Subsequently cases of SARS infections erupted in Hong Kong, Vietnam and Canada, and then spread to over 33 countries and regions. Moreover, SARS erupted in 27 provinces and cities of China, such as Beijing, the Shanxi province, and Mongolia Municipality. The SARS virus is a novel virus, which has strong infectivity and survivability, and high lethality. As of May 31, 2003, 8,360 people worldwide were infected by SARS, and the number of deaths reached 764. Thus, SARS seriously destroyed many people's lives and the economical foundation of the nation.
It has been found by studies that a novel coronavirus is likely to be the cause of SARS. The virus was isolated from the SARS patients and the cadavers of individuals who had been infected with SARS in Hong Kong and Canada, and the anti-hMPV antibody was also found in the serum of some SARS patients. On Apr. 16, 2003, the World Health Organization (WHO) formally confirmed that the SARS virus is the cause of the SARS. In SARS patients, around 90% of the SARS virus infected patients recover spontaneously, and about 10% of the remaining infected patients succumb to the disease. The electron microscope photographs of the respiratory tract slice and the cell culture from the SARS patients show the existence of the coronavirus virosomes. This is a novel type of coronavirus, different from the other known member of the coronavirus genus. This virus can cause a cytopathic effect to green-monkey kidney cells (VERO-E6). The virus replication can be inhibited by the serum recovered from SARS-infected people. And, the immunofluorescene assay (IFA) may be performed using the infected cells and the serum of the recovered people to detect the SARS virus infected cells in the cell culture. This method demonstrates a specific reaction. Studies from the U.S., Canada and Hong Kong show that the serum from non-SARS patients cannot react with this novel coronavirus; the super-antiserums for transmissible gastroenteritis virus (TGEV), murine hepatitis virus (MHV), feline infectious peritonitis virus (FIPV), and 229E human coronavirus may inhibit the growth of the cultured viruses. Virus sequencing conducted in several laboratories show that this novel virus relates to the coronavirus genus, but was different from the other coronavirus groups in the same genus, and is a new variation of the coronavirus. The SARS coronavirus is classified in the coronaviridae strain of nidovirales group of ssRNA positive strand viruses family by species and genus. And, it is a new sub-genus in the coronavirus family.
In March of 2003, scientists discovered the SARS Coronavirus (SARS-CoV), which is the true agent causing SARS, and successfully sequenced the entire SARS-CoV genome. The SARS-CoV genome is comprised of 29727 nucleotides and 11 opening reading frames. The structure of its genome is very similar to other coronaviruses. However, comparison of the genetic history to the sequences shows that the characteristics of SARS-CoV are not completely similar to those of the previously known coronaviruses, and the SARS-CoV has its own specific characteristics in addition to features in common with the other coronaviruses. See Paul A. Rota, M. Steven Oberste, Stephan S. Monroe, W. Allan Nix, Ray Campagnoli, et al. Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science (Sciencexpress) (May 1, 2003); Marco A. Marra, Steven J. M. Jones, Caroline R. Astell, Robert A. Holt, Angela Brooks-Wilson et al., The genomic sequence of the SARS-associated coronavirus, Science 300: 1394-1398 (2003). At the same time, the scientists performed the genome sequencing of 29751 bases of the Toronto Canada isolated strain (Tor2) of SARS-associated coronavirus. The genomic sequence indicated that this coronavirus didn't closely relate to the known coronavirus (including the human coronavirus HcoVOC43, HCoV-229E). The analysis for predicating the genus and species of the viral protein shows that this coronavirus is not closely related to the known three groups of coronaviruses. The genomic sequence will be helpful for people to understand the mechanism of SARS viral infection, diagnose and detect the latent animal host (using PCR method and immunological test), and also be assistant to develop the anti-virus preparation (including neutralizing antibody) and find the antigenic determinants of vaccine (Marco A. Marra, Steven J. M. Jones, Caroline R. Astell, Robert A. Holt, Angela Brooks-Wilson, et al., The genome sequence of the SARS-associated coronavirus, Science, 300:1399-1403 (2003).
Genomic sequencing and bioinformatics analysis has shown that the SARS-CoV virus is mainly comprised of the following functional proteins: a polymerase, a spike (S) glycoprotein, a small envelope (E) protein, a matrix (M) protein, a nucleocapsid (N) protein, and a 3C-like (3CL) protease. Theoretically, all of these proteins can be used as the target of drug design and drug screen for the anti SARS-CoV virus drugs. However, use of 3CL protease as the target in drug designing and screening has some particular advantages: (1) from the functions of 3CL proteases of other coronavirus, it is presumed that the SARS-CoV virus 3CL protease may play an important role during virus replication; (2) there are many 3CL protease inhibitors for other viruses, which can be directly experimented to inhibit the activity of SARS-CoV 3CL protease and in the anti-SARS-CoV test; (3) 3CL protease is very easy to express, so that the protein may be obtained in a short period to screen the drugs. And the screening model at the molecular level in the present invention was constructed just with the expressed SARS-CoV virus 3CL protease by the present inventors, and the protein expression process had been previously described in patent application no. CN 1468961 from the People's Republic of China; (4) SARS-CoV 3CL protease has the higher sequence homology with the main proteinases (Mpro) of the human coronavirus and the genetic gastroenteritis virus, and the 3D-structure of SARS-CoV virus 3CL protease can be constructed with the crystal structures of these two proteases as the templates to perform the design and virtual screening of the inhibitors.
Therefore, the first object of the present invention is to provide a 3D-structure model of SARS-CoV 3CL protease as a target of drug screening for the drugs for treating and/or preventing SARS-CoV viral infection.
The second object of the present invention is to provide a method for screening the drugs for treating and/or preventing SARS-CoV viral infection using the above drug target.
The third object of the present invention is to provide the drugs for treating and/or preventing SARS viral infection screened out using such a drug target.